Methods in molecular biology
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Coronaviruses (CoVs), enveloped positive-sense RNA viruses, are characterized by club-like spikes that project from their surface, an unusually large RNA genome, and a unique replication strategy. CoVs cause a variety of diseases in mammals and birds ranging from enteritis in cows and pigs, and upper respiratory tract and kidney disease in chickens to lethal human respiratory infections. Most recently, the novel coronavirus, SARS-CoV-2, which was first identified in Wuhan, China in December 2019, is the cause of a catastrophic pandemic, COVID-19, with more than 8 million infections diagnosed worldwide by mid-June 2020. Here we provide a brief introduction to CoVs discussing their replication, pathogenicity, and current prevention and treatment strategies. We will also discuss the outbreaks of the highly pathogenic Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV), which are relevant for understanding COVID-19.
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The discovery of induced pluripotent stem cell (iPSC) technology has provided a versatile platform for basic science research and regenerative medicine. With the rise of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) systems and the ease at which they can be utilized for gene editing, creating genetically modified iPSCs has never been more advantageous for studying both organism development and potential clinical applications. However, to better understand the behavior and true therapeutic potential of iPSCs and iPSC-derived cells, a tool for labeling and monitoring these cells in vitro and in vivo is needed. ⋯ The approach involves the integration of the EGFP transgene into the transcriptionally active adeno-associated virus integration site 1 (AAVS1) locus through homology directed repair. The knockin of this transgene results in the generation of iPSC lines with constitutive expression of the EGFP protein that also persists in differentiated iPSCs. These EGFP-labeled iPSC lines are ideal for assessing iPSC differentiation in vitro and evaluating the distribution of iPSC-derived cells in vivo after transplantation into model animals.
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CRISPR Cas9 genome editing allows researchers to modify genes in a multitude of ways including to obtain deletions, epitope-tagged loci, and knock-in mutations. Within 6 years of its initial application, CRISPR-Cas9 genome editing has been widely employed, but disadvantages to this method, such as low modification efficiencies and off-target effects, need careful consideration. Obtaining custom donor vectors can also be expensive and time-consuming. This chapter details strategies to overcome barriers to CRISPR-Cas9 genome editing as well as recent developments in employing this technique.
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Measuring the Replicative Lifespan of Saccharomyces cerevisiae Using the HYAA Microfluidic Platform.
The replicative aging of the budding yeast, Saccharomyces cerevisiae, has been a useful model for dissecting the molecular mechanisms of the aging process. Traditionally, the replicative lifespan (RLS) is measured by manually dissecting mother cells from daughter cells, which is a very tedious process. Since 2012, several microfluidic systems have been developed to automate the dissection process, significantly accelerating RLS determination. Here, we describe a detailed protocol of RLS measurement using a ommercially available microfluidic system based on the HYAA chip design, which enables data collection of up to 8000 cells in a single experiment.
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The third edition of "Plant Proteomics Methods and Protocols," with the title "Advances in Proteomics Techniques, Data Validation, and Integration with Other Classic and -Omics Approaches in the Systems Biology Direction," was conceived as being based on the success of the previous editions, and the continuous advances and improvements in proteomic techniques, equipment, and bioinformatics tools, and their uses in basic and translational plant biology research that has occurred in the past 5 years (in round figures, of around 22,000 publications referenced in WoS, 2000 were devoted to plants). The monograph contains 29 chapters with detailed proteomics protocols commonly employed in plant biology research. They present recent advances at all workflow stages, starting from the laboratory (tissue and cell fractionation, protein extraction, depletion, purification, separation, MS analysis, quantification) and ending on the computer (algorithms for protein identification and quantification, bioinformatics tools for data analysis, databases and repositories). ⋯ Unfortunately, only 10% of them kindly accepted. My gratitude to those who accepted our invitation but also to those who did not, as all of them have contributed to the plant proteomics field. I will enlist, in this introductory chapter, following my own judgment, some of the relevant papers published in the past 5 years, those that have shown us how to enhance and exploit the potential of proteomics in plant biology research, without aiming at giving a too exhaustive list.